The invention relates generally to modified HIV envelope (Env) polypeptides which are useful as immunizing agents or for generating an immune response in a subject, for example a cellular immune response or a protective immune response. More particularly, the invention relates Env polypeptides such as gp120, gp140 or gp160, wherein at least one of the native xcex2-sheet configurations has been modified. The invention also pertains to methods of using these polypeptides to elicit an immune response against a broad range of HIV subtypes.
The human immunodeficiency virus (HIV-1, also referred to as HTLV-III, LAV or HTLV-III/LAV) is the etiological agent of the acquired immune deficiency syndrome (AIDS) and related disorders. (see, e.g., Barre-Sinoussi, et al., (1983) Science 220:868-871; Gallo et al. (1984) Science 224:500-503; Levy et al., (1984) Science 225:840-842; Siegal et al., (1981) N. Engl. J. Med. 305:1439-1444). AIDS patients usually have a long asymptomatic period followed by the progressive degeneration of the immune system and the central nervous system. Replication of the virus is highly regulated, and both latent and lytic infection of the CD4 positive helper subset of T-lymphocytes occur in tissue culture (Zagury et al., (1986) Science 231:850-853). Molecular studies of HIV-1 show that it encodes a number of genes (Ratner et al., (1985) Nature 313:277-284; Sanchez-Pescador et al., (1985) Science 227:484-492), including three structural genesxe2x80x94gag, pol and envxe2x80x94that are common to all retroviruses. Nucleotide sequences from viral genomes of other retroviruses, particularly HIV-2 and simian immunodeficiency viruses, SIV (previously referred to as STLV-III), also contain these structural genes. (Guyader et al., (1987) Nature 326:662-669; Chakrabarti et al., (1987) Nature. 
The envelope protein of HIV-1, HIV-2 and SIV is a glycoprotein of about 160 kd (gp160). During virus infection of the host cell, gp160 is cleaved by host cell proteases to form gp120 and the integral membrane protein, gp41. The gp41 portion is anchored in the membrane bilayer of virion, while the gp120 segment protrudes into the surrounding environment. gp120 and gp41 are more covalently associated and free gp120 can be released from the surface of virions and infected cells.
As depicted in FIG. 1, crystallography studies of the gp 120 core polypeptide indicate that this polypeptide is folded into two major domains having certain emanating structures. The inner domain (inner with respect to the N and C terminus) features a two-helix, two-stranded bundle with a small five-stranded xcex2-sandwich at its termini-proximal end and a projection at the distal end from which the V1/V2 stem emanates. The outer domain is a staked double barrel that lies along side the inner domain so that the outer barrel and inner bundle axes are approximately parallel. Between the distal inner domain and the distal outer domain is a four-stranded bridging sheet which holds a peculiar minidomain in contact with, but distinct from, the inner, the outer domain, and the V1/V2 domain. The bridging sheet is composed of four xcex2-strand structures (xcex2-3, xcex2-2, xcex2-21, xcex2-20, shown in FIG. 1). The bridging region can be seen in FIG. 1 packing primarily over the inner domain, although some surface residues of the outer domain, such as Phe 382, reach into the bridging sheet to form part of its hydrophobic core.
The basic unit of the xcex2-sheet conformation of the bridging sheet region is the xcex2-strand which exists as a less tightly coiled helix, with 2.0 residues per turn. The xcex2-strand conformation is only stable when incorporated into a xcex2-sheet, where hydrogen bonds with close to optimal geometry are formed between the peptide groups on adjacent xcex2-strands; the dipole moments of the strands are also aligned favorably. Side chains from adjacent residues of the same strand protrude from opposite sides of the sheet and do not interact with each other, but have significant interactions with their backbone and with the side chains of neighboring strands. For a general description of xcex2-sheets, see, e.g., T. E. Creighton, Proteins: Structures and Molecular Properties (W. H. Freeman and Company, 1993); and A. L. Lehninger, Biochemistry (Worth Publishers, Inc., 1975).
The gp120 polypeptide is instrumental in mediating entry into the host cell. Recent studies have indicated that binding of CD4 to gp120 induces a conformational change in Env that allows for binding to a co-receptor (e.g, a chemokine receptor) and subsequent entry of the virus into the cell. (Wyatt, R., et al. (1998) Nature 393:705-711; Kwong, P., et al.(1998) Nature 393:648-659). Referring again to FIG. 1, CD4 is bound into a depression formed at the interface of the outer domain, the inner domain and the bridging sheet of gp120.
Immunogenicity of the gp120 polypeptide has also been studied. For example, individuals infected by HIV-1 usually develop antibodies that can neutralize the virus in in vitro assays, and this response is directed primarily against linear neutralizing determinants in the third variable loop of gp120 glycoprotein (Javaherian, K., et al. (1989) Proc. Natl. Acad. Sci. 86:6786-6772; Matsushita, M., et al. (1988) J. Virol. 62:2107-2144; Putney, S., et al. (1986) Science 234:1392-1395; Rushe, J. R., et al. (1988) Proc. Nat. Acad. Sci. USA 85: 3198-3202.). However, these antibodies generally exhibit the ability to neutralize only a limited number of HIV-1 strains (Matthews, T. (1986) Proc. Nat. Acad. Sci. USA. 83:9709-9713; Nara, P. L., et al. (1988) J. Virol. 62:2622-2628; Palker, T. J., et al. (1988) Proc. Natl. Acad. Sci. USA. 85:1932-1936). Later in the course of HIV infection in humans, antibodies capable of neutralizing a wider range of HIV-1 isolates appear (Barre-Sinoussi, F., et al. (1983) Science 220:868-871; Robert-Guroff, M., et al. (1985) Nature (London) 316:72-74; Weis, R., et al. (1985) Nature (London) 316:69-72; Weis, R., et al. (1986) Nature (London) 324:572-575).
Recent work done by Stamatatos et al (1998) AIDS Res Hum Retroviruses 14(13):1129-39, shows that a deletion of the variable region 2 from a HIV-1SF162 virus, which utilizes the CCR-5 co-receptor for virus entry, rendered the virus highly susceptible to serum-mediated neutralization. This V2 deleted virus was also neutralized by sera obtained from patients infected not only with clade B HIV-1 isolates but also with clade A, C, D and F HIV-1 isolates. However, deletion of the variable region 1 had no effect. Deletion of the variable regions 1 and 2 from a LAI isolate HIV-IIIIB also increased the susceptibility to neutralization by monoclonal antibodies whose epitopes are located within the V3 loop, the CD4-binding site, and conserved gp120 regions (Wyatt, R., et al. (1995) J. Virol. 69:5723-5733). Rabbit immunogenicity studies done with the HIV-1 virus with deletions in the V1/V2 and V3 region from the LAI strain, which uses the CXCR4 co-receptor for virus entry, showed no improvement in the ability of Env to raise neutralizing antibodies (Leu et al. (1998) AIDS Res. and Human Retroviruses. 14:151-155).
Further, a subset of the broadly reactive antibodies, found in most infected individuals, interferes with the binding of gp120 and CD4 (Kang, C. -Y., et al. (1991) Proc. Natl. Acad. Sci. USA. 88:6171-6175; McDougal, J. S., et al. (1986) J. Immunol. 137:2937-2944). Other antibodies are believed to bind to the chemokine receptor binding region after CD4 has bound to Env (Thali et al. (1993) J. Virol. 67:3978-3988). The fact that neutralizing antibodies generated during the course of HIV infection do not provide permanent antiviral effect may in part be due to the generation of xe2x80x9cneutralization escapesxe2x80x9d virus mutants and to the general decline in the host immune system associated with pathogenesis. In contrast, the presence of pre-existing neutralizing antibodies upon initial HIV-1 exposure will likely have a protective effect.
It is widely thought that a successful vaccine should be able to induce a strong, broadly neutralizing antibody response against diverse HIV-1 strains (Montefiori and Evans (1999) AIDS Res. Hum. Ret. 15(8):689-698; Bolognesi, D., P., et al. (1994) Ann. Int. Med. 8:603-611; Haynes, B., F., et al. (1996) Science; 271: 324-328.). Neutralizing antibodies, by attaching to the incoming virions, can reduce or even prevent their infectivity for target cells and prevent the cell-to-cell spread of virus in tissue culture (Hu et al. (1992) Science 255:456-459; Burton, D., R. and Montefiori, D. (1997) AIDS 11(suppl. A): 587-598). However as described above, antibodies directed against gp120 do not generally exhibit broad antibody responses against different HIV strains.
Currently, the focus of vaccine development, from the perspective of humoral immunity, is on the neutralization of primary isolates that utilize the CCR5 chemokine co-receptor believed to be important in virus entry (Zhu, T., et al. (1993) Science 261:1179-1181; Fiore, J., et al. (1994) Virology; 204:297-303). These viruses are generally much more resistant to antibody neutralization than T-cell line adapted strains that use the CXCR4 co-receptor, although both can be neutralized in vitro by certain broadly and potent acting monoclonal antibodies, such as IgG1b12, 2G12 and 2F5 (Trkola, A., et al. (1995) J. Virol. 69:6609-6617; D""Sousa P M., et al (1997) J. Infect. Dis. 175:1062-1075). These monoclonal antibodies are directed to the CD4 binding site, a glycosylation site and to the gp41 fusion domain, respectively. The problem that remains, however, is that it is not known how to induce antibodies of the appropriate specificity by vaccination. Antibodies (Abs) elicited by gp120 glycoprotein from a given isolate are usually only able to neutralize closely related viruses generally from similar, usually from the same, HIV-1 subtype.
Despite the above approaches, there remains a need for Env antigens that can elicit an immunological response (e.g., neutralizing and/or protective antibodies) in a subject against multiple HIV strains and subtypes, for example when administered as a vaccine. The present invention solves these and other problems by providing modified Env polypeptides (e.g., gp120) to expose epitopes in or near the CD4 binding site.
In accordance with the present invention, modified HIV Env polypeptides are provided. In particular, deletions and/or mutations are made in one or more of the 4-xcex2 antiparallel-bridging sheet in the HIV Env polypeptide. In this way, enough structure is left to allow correct folding of the polypeptide, for example of gp120, yet enough of the bridging sheet is removed to expose the CD4 groove, allowing an immune response to be generated against epitopes in or near the CD4 binding site of the Env polypeptide (e.g., gp120).
In one aspect, the invention includes a polynucleotide encoding a modified HIV Env polypeptide wherein the polypeptide has at least one modified (e.g., deleted or replaced) amino acid residue deleted in the region corresponding to residues 421 to 436 relative to HXB-2, for example the constructs depicted in FIGS. 6-29 (SEQ ID NOs:3 to 26). In certain embodiments, the polynucleotide also has the region corresponding to residues 124-198 of the polypeptide HXB-2 (e.g., V1/V2) deleted and at least one amino acid deleted or replaced in the regions corresponding to the residues 119 to 123 and 199 to 210, relative to HXB-2. In other embodiments, these polynucleotides encode Env polypeptides having at least one amino acid of the small loop of the bridging sheet (e.g., amino acid residues 427 to 429 relative to HXB-2) deleted or replaced. The amino acid sequences of the modified polypeptides encoded by the polynucleotides of the present invention can be based on any HIV variant, for example SF162.
In another aspect, the invention includes immunogenic modified HIV Env polypeptides having at least one modified (e.g., deleted or replaced) amino acid residue deleted in the region corresponding to residues 421 to 436 relative to HXB-2, for example a deletion or replacement of one amino acids in the small loop region (e.g., amino acid residues 427 to 429 relative to HXB-2). These polypeptides may have modifications (e.g., a deletion or a replacement) of at least one amino acid between about amino acid residue 420 and amino acid residue 436, relative to HXB-2 and, optionally, may have deletions or truncations of the V1 and/or V2 regions. The immunogenic, modified polypeptides of the present invention can be based on any HIV variant, for example SF162.
In another aspect, the invention includes a vaccine composition comprising any of the polynucleotides encoding modified Env polypeptides described above. Vaccine compositions comprising the modified Env polypeptides and, optionally, an adjuvant are also included in the invention.
In yet another aspect, the invention includes a method of inducing an immune response in subject comprising, administering one or more of the polynucleotides or constructs described above in an amount sufficient to induce an immune response in the subject. In certain embodiments, the method further comprises administering an adjuvant to the subject.
In another aspect, the invention includes a method of inducing an immune response in a subject comprising administering a composition comprising any of the modified Env polypeptides described above and an adjuvant. The composition is administered in an amount sufficient to induce an immune response in the subject.
In another aspect, the invention includes a method of inducing an immune response in a subject comprising
(a) administering a first composition comprising any of the polynucleotides described above in a priming step and
(b) administering a second composition comprising any of the modified Env polypeptides described above, as a booster, in an amount sufficient to induce an immune response in the subject. In certain embodiments, the first composition, the second composition or both the first and second compositions further comprise an adjuvant.
These and other embodiments of the subject invention will readily occur to those of skill in the art in light of the disclosure herein.